Alexander Varshavsky

Alexander J. Varshavsky (born in 1946, in Moscow, Russia) is a Russian-American biochemist and geneticist. He works at Caltech as the Morgan Professor of Biology. In the year 1977, Varshavsky left Russia and immigrated to United States.

His laboratory, initially at the Massachusetts Institute of Technology, and later at Caltech, has discovered, during the 1980s, degrons in the short-lived proteins and ubiquitin system biological fundamentals. His current research continues to focus on the ubiquitin system and N-degron pathways.

Contributions in the ubiquitin field

In 1986, the Varshavsky laboratory discovered and analyzed the degrons in short-lived proteins.^[1][2][3] “Degron”, by now a standard term, was introduced by Varshavsky in 1991. In 1984-1990, the Varshavsky lab discovered biological fundamentals of ubiquitin system.^[2][3][4] The field of ubiquitin and regulated protein degradation was created in the 1980s through discoveries, during 19781990, that revealed three sets of previously unknown facts. The first set of these facts (item 1 below) was discovered by A. Hershko (Technion, Israel) laboratory.^[5] The other two sets (items 2 and 3 below) were discovered by the Varshavsky laboratory, then at the Massachusetts Institute of Technology (Cambridge, Massachusetts).^[2][3][4]

(1) A. Ciechanover and A. Hershko demonstrated that ubiquitin, a 76-residue protein, is covalently conjugated to other proteins in cell extracts, a novel protein modification involved in the ATP-dependent protein breakdown in extracts from mammalian reticulocytes.^[5] Ubiquitylation of a test protein in a reticulocyte extract caused it to become short-lived in the extract. Hershko, Ciechanover and their colleagues also discovered that ubiquitin-protein conjugation is mediated by a cascade of enzymes, termed E1, E2, & E3. The studies were carried out using cell-free (in vitro) extracts and isolated E1-E3 enzymes.^[5] At that time, in the early 1980s, physiological significance of the ubiquitin system and its specific biological functions remained unknown.

(2) In 1986, the in vivo selectivity of ubiquitylation (ubiquitin-protein conjugation) was shown, by the Varshavsky lab, to be determined by degradation signals (degrons) in cellular proteins.^[1][2][3][4] N-terminal degrons, called N-degrons, were the first degradation signals to be discovered. Ubiquitindependent proteolytic systems that selectively destroy proteins bearing N-degrons are called N-degron pathways. Prior to 2019, these were referred N-end rule pathways.^[3][4]

(3) During 1984-1990, the Varshavsky lab discovered that ubiquitylation has remarkably broad biological functions, to a large extent through control of the in vivo levels of cellular proteins.^{[1][2][3][4][6][7]} Varshavsky and others demonstrated in 1984 that the majority of protein breakdown in vivo, or in living cells, requires ubiquitylation. Subsequently, the initial distinct roles of ubiquitylation were identified, which included DNA repair (1987), the cell division cycle (1988), stress responses (1987), protein synthesis (1989), and transcriptional control (1990).^{[2][3][4][6][7]} Additionally, the Varshavsky lab discovered the MATalpha2 repressor in 1990, cloned the first genes encoding mature ubiquitin precursors (1984–1989), identified the first ubiquitin-conjugating (E2) enzymes that have precise biological features (1987–1988), discovered a nonproteolytic ubiquitin fetaure (1989), discovered the first E3 ubiquitin ligase and cloned it in 1990, and cloning the first deubiquitylating enzymes, referred to as UBP1–UBP3.ns!.^{[1][2][3][4][6][7]} More than 600 different E3 ubiquitin ligases have been found to be encoded in the human genome, according to subsequent research, which was made possible by the 1990 cloning of the initial E3 ubiquitin ligase, known as UBR1. The ubiquitin system's enormous functional range is supported by this multitude of E3s. Additionally, In 1989, the Varshavsky lab found the first specific substrate linked polyubiquitin chains and demonstrated in 1990 that the ubiquitin system's ability to degrade proteins is subunit-selective, meaning that it can specifically destroy an oligomeric protein's subunit to allow for protein remodeling.(references ^{[1][2][3][4][6][7]} and references therein).

The initial discovery of degrons and the biological principles underlying the ubiquitin system were made possible by the advancements outlined in items 2 and 3.^[5][8][1][2] The 1980s saw complementary discoveries made by the laboratories of Hershko and Varshavsky (see items 1-3 above), which led to the development of the modern paradigm that emphasizes the critical role that regulated proteolysis plays in controlling the levels of particular proteins in vivo as opposed to transcription and protein synthesis. These discoveries showed that the traditional transcription-and translation-based regulation is often not only comparable but even more significant than the control achieved through protein degradation. Given the wide range of functions of the ubiquitin system and the numerous ways in which ubiquitin-dependent processes can cause malfunctioning in disease or during aging, from cancer and neurodegenerative syndromes to immune system perturbations and numerous other illnesses, including birth defects, the resulting change in our understanding of biological circuits has significant implications for medicine.^{[5][8][2][3][4]}

Varshavsky's colleagues carried out further research on the ubiquitin system in the ensuing decades (from 1990 to the present), focusing primarily on N-degron pathways. Selective misfolded protein degradation, sensing of molecules like oxygen, heme, short peptides and nitric oxide, control over DNA transcription, replication, repair, and chromosome cohesion/segregation, control over peptide transport, meiosis, chaperones, cytoskeletal proteins, gluconeogenesis, autophagy, apoptosis, adaptive & innate immunity that includes inflammation, cardiovascular development, neurogenesis, spermatogenesis, and circadian rhythms; diverse involvements in human diseases such as cancer, neurodegeneration, and defects of immunity; a variety of roles in bacteria; and many functions in plants, including seed germination and oxygen/NO sensing (references ^{[8][2][3][6][7][9][10]} and references therein).

Contributions outside the ubiquitin field

• The discovery, in 1978-1979, of the first nucleosome-depleted, nuclease-hypersensitive regions in chromosomes. Such “exposed” chromosomal regions are characteristic of transcriptional promoters, recombination hotspots, and the origination of the DNA replication.^[1][2]
• The discovery, in 1980-1981, of the initial chromosomal cohesion/segregation pathway. It involves the formation, during DNA replication, of multiply intertwined (multicatenated) sister chromatids, and their later stepwise decatenation by type-2 DNA topoisomerases.^[1][2]
• The idea, in 2007, that DNA deletions (and less frequent insertions) that are characteristic of cancer cells, can be used as irreversibly present (non-reverting) cancer-specific signposts, thereby making possible a selective therapy of cancers that would be impervious to tumor progression.^[11][12]
• The sleep-cause hypothesis as proposed by fragment generation (FG) between 2012 and 2019.^[13] In this conjecture, a molecular cause of sleep stems from production, during wakefulness, of numerous extracellular and intracellular protein-sized protein fragments that can be transiently beneficial but can also perturb, through their diverse and cumulative effects, the functioning of the brain and other organs. The FG hypothesis posits that sleep evolved, at least in part, to counteract overproduction (owing to an insufficiently fast elimination) of hundreds of different protein fragments during wakefulness.^[13] The FG hypothesis is consistent with available experimental evidence. It remains to be verified.
• Inventions of genetic and biochemical methods (1980-2017) (see references^[1][2][4] and references therein):

(i) Two-dimensional electrophoretic mapping of DNA replication/multicatenation intermediates, in 1980-1981.

(ii) Nucleosome mapping using two-dimensional hybridization in 1982.

(iii) The 1986 ubiquitin fusion technique. This method makes it possible to expose, in vivo, a desired N-terminal residue in a protein of interest. Owing to the design of the genetic code, all nascent proteins bear the N-terminal Met residue, which is either retained in or removed from mature proteins. The ubiquitin fusion technique makes it possible to “bypass” the endogenous rules of N-terminal Met removal.

(iv)The Chromatin Immunoprecipitation Assay, 1988. Advanced versions of ChIP are being used for mapping in vivo locations of chromosomal proteins.

(v) Hypersensitivity to D2O was identified as a conditional phenotype that was generally applicable. (1988)

(vi) Heat-activated N-degron, in 1994, for producing temperature-sensitive mutants.

(vii) Split-ubiquitin method, in 1994, for detecting protein interactions in vivo. The central idea of the split-ubiquitin technique opened the field of single subunit split proteins, such as split-GFP, split lactamase, split Cas9 CRISPR nuclease, and many other split protein sensors and effectors.

(viii) Ubiquitin translocation assay, in 1994, for analyzing, in vivo, mechanisms and kinetics of protein translocation across specific cellular membranes.

(ix) Ubiquitin sandwich technique, in 2000, which uses ubiquitin fusions and distinct tandem reporters to detect and measure cotranslational proteolysis in vivo.

(x) The subunit decoy technique was developed in 2013 to examine how steric shielding of conditional degrons regulates subunit stoichiometries in the oligomeric proteins in vivo.

In 2017, Promoter Reference Technique was introduced. It is a protein degradation assay that uses RNA aptamers as a reference and eliminates the need for global translation inhibitors.

References

1. Varshavsky, Alexander (2008). "Discovery of Cellular Regulation by Protein Degradation". Journal of Biological Chemistry. 283 (50): 34469–34489. doi:10.1074/jbc.x800009200. ISSN 0021-9258. PMC 3259866. PMID 18708349.
2. Varshavsky, A. "(2022) interview about life and work, to David Zierler, Caltech Heritage Project" (PDF). Caltech.
3. Bachmair, Andreas; Finley, Daniel; Varshavsky, Alexander (1986-10-10). "In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue". Science. 234 (4773): 179–186. doi:10.1126/science.3018930. ISSN 0036-8075.
4. Varshavsky, Alexander (2019-01-08). "N-degron and C-degron pathways of protein degradation". Proceedings of the National Academy of Sciences. 116 (2): 358–366. doi:10.1073/pnas.1816596116. ISSN 0027-8424. PMC 6329975. PMID 30622213.
5. Hershko, Avram; Ciechanover, Aaron; Varshavsky, Alexander (2000). "The ubiquitin system". Nature Medicine. 6 (10): 1073–1081. doi:10.1038/80384. ISSN 1078-8956.
6. Jentsch, Stefan; McGrath, John P.; Varshavsky, Alexander (1987). "The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme". Nature. 329 (6135): 131–134. doi:10.1038/329131a0. ISSN 1476-4687.
7. Johnson, Erica S.; Gonda, David K.; Varshavsky, Alexander (1990). "Cis-trans recognition and subunit-specific degradation of short-lived proteins". Nature. 346 (6281): 287–291. doi:10.1038/346287a0. ISSN 1476-4687.
8. Varshavsky, Alexander (2014). "Discovery of the Biology of the Ubiquitin System". JAMA. 311 (19): 1969. doi:10.1001/jama.2014.5549. Retrieved 2023-10-05.
9. Oh, Jang-Hyun; Hyun, Ju-Yeon; Chen, Shun-Jia; Varshavsky, Alexander (2020-05-19). "Five enzymes of the Arg/N-degron pathway form a targeting complex: The concept of superchanneling". Proceedings of the National Academy of Sciences. 117 (20): 10778–10788. doi:10.1073/pnas.2003043117. ISSN 0027-8424. PMC 7245096. PMID 32366662.
10. Vu, Tri T. M.; Mitchell, Dylan C.; Gygi, Steven P.; Varshavsky, Alexander (2020-12-08). "The Arg/N-degron pathway targets transcription factors and regulates specific genes". Proceedings of the National Academy of Sciences. 117 (49): 31094–31104. doi:10.1073/pnas.2020124117. ISSN 0027-8424. PMC 7733807. PMID 33229537.
11. Varshavsky, Alexander (2007-09-18). "Targeting the absence: Homozygous DNA deletions as immutable signposts for cancer therapy". Proceedings of the National Academy of Sciences. 104 (38): 14935–14940. doi:10.1073/pnas.0706546104. ISSN 0027-8424. PMC 1986591. PMID 17846424.
12. Varshavsky, Alexander; Lewis, Kim; Chen, Shun‐Jia (2023). "Deletions of DNA in cancer and their possible uses for therapy". BioEssays. 45 (7). doi:10.1002/bies.202300051. ISSN 0265-9247.
13. Varshavsky, Alexander (2019-05-28). "On the cause of sleep: Protein fragments, the concept of sentinels, and links to epilepsy". Proceedings of the National Academy of Sciences. 116 (22): 10773–10782. doi:10.1073/pnas.1904709116. ISSN 0027-8424. PMC 6561186. PMID 31085645.

Notes